Cross-linked enzyme aggregates and entrapment immobilization of maltogenic amylase from Bacillus lehensis G1 for Maltooligosaccharides synthesis

Maltooligosaccharides (MOS) are potential oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). Although MOS can be synthesized by using free enzymes, this process is hampered by poor enzyme recovery and lack of enzyme stability, which makes it unrealistic for applications. To overcome these drawbacks, several optimization methods, including enzyme immobilization approach, could be applied. A carrier-free immobilization technique that uses cross-linked enzyme aggregates (CLEAs) enhances the stability of the enzymes. Indeed, a decrease in substrate accessibility of CLEAs may hinder CLEAs applications. The substrate accessibility problem of CLEAs formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs have particles that are small in size, soft and mechanically unstable, which can cause enzyme leaching and reduce the activity, as well as the performance of the enzyme. To address these problems, p-CLEAs were entrapped in calcium alginate beads (CA). In this study, a formation of cross-linked enzyme aggregates of Mag1 (Mag1-CLEAs) were carried out to improve the stability and reusability of free Mag1. The substrate accessibility problem of Mag1-CLEAs was solved by the formation of porous CLEAs of Mag1 (Mag1-p-CLEAs). All factors affecting the formation of CLEAs were investigated. The highest activity recovery of Mag1-CLEAs 58.14 % (18.6 U/mg) was obtained at 80 % (w/v) ammonium sulphate (precipitant), 0.25 % (w/v) chitosan (cross linker) with cross-linking time of 1.5 h. In comparison, Mag1-p-CLEAs prepared with 0.8 % (w/v) citrus pectin (porous agent) exhibited 91.20 % (29.2 U/mg) activity. This developed porous material exhibited larger particles size (1.60 µm) and pore size distribution of 8 – 1000 nm. Mag1-p-CLEAs noticeably retained 80 % of their activity after 30 min of incubation at 40 °C and showed longer half-life compared to free Mag1 and Mag1-CLEAs. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin (ß-CD) accessibility. Next, Mag1-p-CLEAs were entrapped into calcium alginate beads. Mag1-p-CLEAs-CA prepared with 2.5 % (w/v) sodium alginate and 0.6 % (w/v) calcium chloride yielded 53.16 % (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards ß-CD (Km = 0.62 mM). MOS (261.9 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C through hydrolysis reaction of ß-CD. Although, Mag1-p-CLEAs-CA have low transglycosylation activity, they have superior reusability and can maintain their activity for up to 11 cycles. In conclusion, the combination of CLEAs technology with entrapment approach, has proven to be a promising tool to develop stable enzymes. The developed Mag1-p-CLEAs-CA are potential biocatalyst for the continuous production of MOS